Fluorescein disodium

Zein (CAS number 9010-66-62, EC number: 232-722-9), rhodamine B, disodium fluorescein, bromophenol blue, methylene blue, and ethanol were purchased from Sigma-Aldrich (Milan, Italy). All other materials and solvents used in this investigation were of analytical grade (Carlo Erba, Milan, Italy), while cellulose membrane Spectra/Por MWCO 50 kDa was obtained from Spectrum Laboratories Inc. (Eindhoven, The Netherlands).

The Pterygoplicgthys samples are supplied by the University of Michoacan of San Nicolas de Hidalgo (Morelia, Mexico). The enzymes were supplied by ENMEX (Tlalnepantla de Baz, Mexico): two neutrases, HT Proteolitic ^® L200 (HT), and Proteasa Fungal (PF) and one alcalase, PAL^®660 (PAL). The following chemicals were purchased from Sigma Aldrich^® (Gillingham, UK): Bis-tris Propane (B6755); O-phthaldialdehyde (OPA, P1378); Sodium-dodecyl-sulphate (SDS) (L3771); Dithiothreitol 99% (DTT, D0632); Potassium persulfate (216224) was purchased from Sigma Aldrich^®; 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, A1888); 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ, T1253); Ferric Chloride Hexahydrate (F2877); Sodium Acetate Trihydrate (S8625); Glacial Acetic Acid (320099); Disodium fluorescein (F6377); 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH, 440914); Sodium chloride [NaCl] > 99.5% (S7653); Tris (hydroxymethyl) methylamine (T1503); Angiotensin-converting enzyme (ACE, A6778; Hydrochloric acid 36.5–38.0%, (H1758); FAPGG—N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly ≥98% (F7131); Glycerol solution 86–89% (49781); Trichloroacetic acid (TCA, T6399).

Reagents were acquired from various suppliers: authentic standards of kuromanin (cyanidin 3-O-glucoside chloride), rutin (quercetin 3-O-rutinoside), chlorogenic acid (5-caffeoylquinic acid) from Extrasynthèse (Genay, France); gallic acid, Folin–Ciocalteu phenol reagent, Trolox [(S)-(-)-6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid], fluorescein disodium, ABTS [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], AAPH [2,2′-azobis (2-methyl-propionamide)], acetonitrile, formic acid, ethanol, and organic acids (all HPLC grade) from Sigma-Aldrich (St. Louis, MO, USA). ICP-AES standards were obtained from Ultra Scientific Analytical Solutions (Bologna, Italy). Milli-Q water was used (Merck Millipore, Darmstadt, Germany).

For the analysis of ORAC, the method of Ou et al. was used, where a 25 μL of hydrophilic extract, 50 times diluted, was added to the microplates together with 150 μL of fluorescein (81 nmol/L) and 25 μL of 2,2’-azobis (2-amidino-propane) dihydrochloride (AAPH) (152 mmol/L); AAPH and fluorescein disodium were purchased from Sigma-Aldrich Chemical Company (Steinheim, Germany). The plates were incubated in a microplate reader (Hidex Sense) at 37°C, and readings were taken every 1 min for a period of 90 min, using the fluorescence detector with 485 nm excitation wavelength and emission at 528 nm (24 (link)). A standard Trolox curve was drawn on each test microplate.

Reagents were purchased from various suppliers as follows: Authentic standards of kuromanin (cyanidin 3-O-glucoside chloride), chlorogenic acid (3-caffeoylquinic acid), gentisic acid, rutin (quercetin 3-O-rutinoside) (Extrasynthèse, Genay, France); the standard for carotenoids (lutein, carotene, α-carotene, β-carotene and lycopene) were purchased from CaroteNature (Lupsingen, Switzerland); gallic acid, Folin-Ciocalteu's phenol reagent, Trolox [(S)-(-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], ABTS [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], fluorescein disodium, AAPH [2,2′-azobis (2-methyl-propionamide)], ascorbic acid, meta-phosphoric acid, DTT (dithiothreitol), BHT (butylated hydroxytoluene) as well as acetonitrile, ethanol, methanol, acetone, methyl tert-butyl ether, formic acid and acetic acid (all HPLC grade) (Sigma-Aldrich, St. Louis, MO, USA). In all experiments Milli-Q (Merck Millipore, Darmstadt, Germany) water was used.

Reagents were acquired from various suppliers: cyanidin 3-O-glucoside chloride, rutin (quercetin 3-O-rutinoside), and chlorogenic acid (5-caffeoylquinic acid) were purchased from Extrasynthèse (Genay, France); gallic acid, Folin–Ciocalteu phenol reagent, Trolox ((S)-(-)-6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid), fluorescein disodium, ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), AAPH (2,2′-azobis (2-methyl-propionamide)), acetonitrile, formic acid, ethanol, and organic acids (all HPLC-grade) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q water was used (Merck Millipore, Darmstadt, Germany).

2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 6-hydroxy-2,5,7,8-tetramethylchromium-2-carboxylic acid (Trolox), fluorescein disodium (FL), 2,2′-azobis (2-amidinopropane dihydrochloride) (AAPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide (MTT), dimethyl sulfoxide (DMSO), and 2′7′-dichlorofluorescein diacetate (DCFA-DA) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Lipopolysaccharide (LPS) was purchased from Merck (Kenilworth, NJ, USA). All other reagents were of analytical grade.
The soybean protein isolate (PI) was provided by Emilio Peña Aditivos Alimentarios S.A. (Valencia, Spain). The lunasin peptide was synthesized by DGpeptides Co., Ltd. (Hangzhou, China) with a purity of 95.2%.